Optimization of the electrokinetic supercharging preconcentration for high-sensitivity microchip gel electrophoresis on a cross-geometry microchip

We developed a novel on-line preconcentration procedure for microchip gel electrophoresis (MCGE), which enables application of electrokinetic supercharging (EKS) for highly sensitive detection of DNA fragments on a cross-geometry microchip. In comparison with conventional pinched injection using the cross microchip, the present approach allows loading a much larger amount of the sample by taking advantage of a newly developed operational mode. In order to obtain high preconcentration effect and prevent splitting of an enriched sample into subchannels, i.e., off the detector range, effects of the voltage applied on the reservoirs and the time of isotachophoretic preconcentration were examined. The optimal balance between the voltage and time was found for a high-sensitivity analysis of DNA fragments. After experimental optimization the detection limit of a 150 bp fragment was as low as 0.22 mg/L (S/N = 3) that is 10 times better than using the conventional pinched injection.     

基于微芯片电泳的脱氧核糖核酸片段的浓缩和分离

采用超负荷电动供给(electrokinetic supercharging, EKS)预浓缩技术,在微芯片电泳(MCE)上对脱氧核糖核酸（DNA）片段进行浓缩和分离。EKS是集合样品电动进样(EKI)和过渡等速电泳(tITP)的一种在线浓缩方法。研究表明：采用该方法后,在40.5 mm长的单通道芯片上能够实现对低浓度样品的大量进样、浓缩和基线分离。在普通的紫外检测条件(检测波长为260 nm)下,对DNA片段的平均检出限(S/N=3)约为0.07 mg/L,仅为十字芯片上的微芯片电泳检出限的1/40。本文还对浓缩过程中的一些关键因素和定性分析进行了探讨。     

Membrane-mediated ultrafast restriction digestion and subsequent rapid gel microchip electrophoresis of DNA

Ultrafast, membrane-mediated restriction digestion of DNA molecules followed by rapid gel microchip electrophoresis of the resulting fragments is described. Combination of restriction endonuclease digestion on small pore-size microfibrous membranes with sample loading and electrophoresis analysis in a multilane (up to 96) format resulted in very fast restriction digest based microscale DNA analysis. Complete digestion of several nanogram target DNA was accomplished on the microporous membrane at room temperature just in a few minutes with a single or a combination of various restriction enzymes, using only submicroliter quantities of samples and reagents. The reaction mixture containing membrane also served as sample loading device for the subsequent gel microchip electrophoresis based analysis. This work establishes methods for high-speed, high-throughput DNA analysis, featuring extremely low sample and reagent consumption, and fast restriction digestion in combination with sample loading and rapid gel microchip analysis of the resulting fragments. The entire restriction digestion, sample loading and electrophoresis analysis process required less than 20 min.     

High-sensitivity capillary gel electrophoretic analysis of DNA fragments on an electrophoresis microchip using electrokinetic injection with transient isotachophoretic preconcentration

The research adopted a single-channel microchip as the probe, and focused electrokinetic injection combined with transient isotachophoresis preconcentration technique on capillary electrophoresis microchip to improve the analytical sensitivity of DNA fragments. The channel length, channel width and channel depth of the used microchip were 40.5 mm, and 110 and 50 μm, respectively. The separation was detected by CCD (charge-coupled device) (effective LENGTH=25 mm, 260 nm). A 1/100 diluted sample (0.2 mg/l of each DNA fragment) of commercially available stepladder DNA sample could be baseline separated in 120 s with S/N=2–5. Compared with conventional chip gel electrophoresis, the proposed method is ideally suited to improve the sensitivity of DNA analysis by chip electrophoresis.     

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.     

Fast parallel detection of feline panleukopenia virus DNA by multi‐channel microchip electrophoresis with programmed step electric field strength

A multi‐channel microchip electrophoresis using a programmed step electric field strength (PSEFS) method was investigated for fast parallel detection of feline panleukopenia virus (FPV) DNA. An expanded laser beam, a 10× objective lens, and a charge‐coupled device camera were used to simultaneously detect the separations in three parallel channels using laser‐induced fluorescence detection. The parallel separations of a 100‐bp DNA ladder were demonstrated on the system using a sieving gel matrix of 0.5% poly(ethylene oxide) (M_r = 8 000 000) in the individual channels. In addition, the PSEFS method was also applied for faster DNA separation without loss of resolving power. A DNA size marker, FPV DNA sample, and a negative control were simultaneously analyzed with single‐run and one‐step detection. The FPV DNA was clearly distinguished within 30 s, which was more than 100 times faster than with conventional slab gel electrophoresis. The proposed multi‐channel microchip electrophoresis with PSEFS was demonstrated to be a simple and powerful diagnostic method to analyze multiple disease‐related DNA fragments in parallel with high speed, throughput, and accuracy.     

Impact of reservoir potentials on the analyte behavior in microchip electrophoresis: computer simulation and experimental validation for DNA fragments

Fundamental understanding of the impact of reservoir potentials on the analyte behavior on the microfluidic chips is an important issue in microchip electrophoresis (MCE) for suitable injection and separation of analytes, since the applied potentials may significantly affect the shape of sample plug, sample leakage from the injection channel to the separation channel, injected sample amount, and separation efficiency. This study addressed this issue for the case of a conventional cross-geometry microchip with four reservoirs using computer simulations, the results of which were verified by the analysis of DNA fragments. For the microchip with a definite structure and migration distance, the injected sample amount was shown to be the vital parameter for improving the limit of detection and resolution. During injection, the shape of the sample plug could be adjusted by varying the reservoir potentials. It was demonstrated that a "magnified injection" (applying high voltage on the three reservoirs to the sample reservoir) is useful to enhance the detection sensitivity depending on the analyte composition, although such injection was previously avoided because of introducing too large amounts of the analyte in comparison with two established modes, floating and pinched injection. Optimal magnified injection was proved to improve the sensitivity for about 4 times over that of pinched injection for the analysis of DNA step ladders using microchip gel electrophoresis (MCGE). Sample leakage of DNA fragments could be suppressed by applying a high positive voltage on injection channel during separation, but the voltage degraded the injected amount and resolution.     

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.     

Microchip electrophoresis of DNA following preconcentration at photopatterned gel membranes

Rapid separation of nucleic acids by microchip electrophoresis could streamline many biological applications, but conventional chip injection strategies offer limited sample stacking, and thus limited sensitivity of detection. We demonstrate the use of photopatterned polyacrylamide membranes in a glass microfluidic device, with or without fixed negative charges, for preconcentration of double-stranded DNA prior to electrophoretic separation to enhance detection limits. We compared performance of the two membrane formulations (neutral or negatively charged) as a function of DNA fragment size, preconcentration time, and preconcentration field strength, with the intent of optimizing preconcentration performance without degrading the subsequent electrophoretic separation. Little size-dependent bias was observed for either membrane formulation when concentrating dsDNA > 100 bp in length, while the negatively charged membrane more effectively blocks passage of single-stranded oligonucleotide DNA (20-mer ssDNA). Baseline resolution of a six-band dye-labeled ladder with fragments 100-2000 bp in size was obtained in <120 s of separation time, with peak efficiencies in the range of 2000-15,000 plates/cm, and detection limits as low as 1 pM per single dye-labeled fragment. The degree of preconcentration is tunable by at least 49-fold, although the efficiency of preconcentration was found to have diminishing returns at high field and/or long times. The neutral membrane was found to be more robust than the negatively charged membrane, with approximately 2.5-fold larger peak area during the subsequent separation, and less decrease in resolution upon increasing the preconcentration field strength.     

On-channel base stacking in microchip capillary gel electrophoresis for high-sensitivity DNA fragment analysis

We evaluated a novel strategy for high-sensitivity DNA fragment analysis in a conventional glass double-T microfluidic chip. The microchip allows for a DNA on-channel concentration based on base stacking (BS) with a microchip capillary gel electrophoretic (MCGE) separation step in a poly(vinylpyrrolidone) (PVP) sieving matrix. Depending if low conductivity caused a neutralization reaction between the hydroxide ions and the run buffer component Tris+, the stacking of DNA fragments were processed in the microchip. Compared to a conventional MCGE separation with a normal electrokinetic injection, the peak heights of 50-2650-base pair (bp) DNA fragments on the MCGE-BS separation were increased 3.9-8.0-fold. When we applied the MCGE-BS method to the analysis of a clinical sample of bovine theileria after PCR reaction, the peak height intensity of the amplified 816-bp DNA fragment from the 18S rRNA of T. buffeli was enhanced 7.0-fold compared to that of the normal injection method.     

Modification of a poly(methyl methacrylate) injection-molded microchip and its use for high performance analysis of DNA

Inexpensive and permanently modified poly(methyl methacrylate)(PMMA) microchips were fabricated by an injection-molding process. A novel sealing method for plastic microchips at room temperature was introduced. Run-to-run and chip-to-chip reproducibility was good, with relative standard deviation values between 1-3% for the run-to-run and less than 2.1% for the chip-to-chip comparisons. Acrylonitrile-butadiene-styrene (ABS) was used as an additive in PMMA substrates. The proportions of PMMA and ABS were optimized. ABS may be considered as a modifier, which obviously improved some characteristics of the microchip, such as the hydrophilicity and the electro-osmotic flow (EOF). The detection limit of Rhodamine 6G dye for the modified microchip on the home-made microchip analyzer showed a dramatic 100-fold improvement over that for the unmodified PMMA chip. A detection limit of the order of 10(-20) mole has been achieved for each injected psiX-174/HaeIII DNA fragment with the baseline separation between 271 and 281 bp, and fast separation of 11 DNA restriction fragments within 180 seconds. Analysis of a PCR product from the tobacco ACT gene was performed on the modified microchip as an application example.     

Fabrication of SU-8 based microchip electrophoresis with integrated electrochemical detection for neurotransmitters

A new SU-8 based microchip capillary electrophoresis (MCE) device has been developed for the first time with integrated electrochemical detection. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology has allowed the monolithic integration in the device of the electrochemical detection system based on platinum electrodes. The fabrication of the chips presented in this work is totally compatible with reel-to-reel techniques, which guarantee a low cost and high reliability production. The influence of relevant experimental variables, such as the separation voltage and detection potential, has been studied on the SU-8 microchip with an attractive analytical performance. Thus, the effective electrical isolation of the end-channel amperometric detector has been also demonstrated. The good performance of the SU-8 device has been proven for separation and detection of the neurotransmitters, dopamine (DA) and epinephrine (EP). High efficiency (30,000-80,000 N/m), excellent precision, good detection limit (450 nM) and resolution (0.90-1.30) has been achieved on the SU-8 microchip. These SU-8 devices have shown a better performance than commercial Topas (thermoplastic olefin polymer of amorphous structure) microchips. The low cost and versatile SU-8 microchip with integrated platinum film electrochemical detector holds great promise for high-volume production of disposable microfluidic analytical devices.     

Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments

Aiming to achieve high-performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross-geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP-stacked zones passed the cross-part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross-part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD-ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross-microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single-channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection.     

Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification

Optimal detection of a pathogen present in biological samples depends on the ability to extract DNA molecules rapidly and efficiently. In this paper, we report a novel method for efficient DNA extraction and subsequent real-time detection in a single microchip by combining laser irradiation and magnetic beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli and Gram-positive bacterial cells as well as the hepatitis B virus mixed with human serum. We further demonstrate that the real-time pathogen detection was performed with pre-mixed PCR reagents in a real-time PCR machine using the same microchip, after laser irradiation in a hand-held device equipped with a small laser diode. These results suggest that the new sample preparation method is well suited to be integrated into lab-on-a-chip application of the pathogen detection system.     

通用型激光诱导荧光微流控芯片分析仪的研制与性能考察

设计和研制了一种通用型激光诱导荧光微流控芯片分析仪.检测部分按共聚焦检测原理设计,采用CCD(电荷耦合器件)监测通道,三维自动调节聚焦,发射波长滤光片可方便地更换以适应多种染料选择,能分别显示进样和分离通道2条电流-时间曲线.考察了该分析仪的检测灵敏度、检测极限和线性范围,显示了分析灵敏度高,检测限低和线性范围宽等特点,在自制注塑型PMMA塑料芯片上实现了φX174Haedi-gesT_dNA片段的分离测定和烟叶act基因PCR产物的分析     

A study on the system of nonaqueous microchip electrophoresis with on‐line peroxyoxalate chemiluminescence detection

This article describes a further development of our previously reported miniaturized analysis system of microchip electrophoresis with on‐line chemiluminescence detection. The system, developed first time for nonaqueous microchip electrophoresis with peroxyoxalate chemiluminescence detection, consists of a suction pressure device for sample or reagent introduction, a constant voltage supplied for electrophoretic separation, an either hydrophilic or hydrophobic porous polymer plug for preventing chemiluminescence reagent flowing upstream and a spiral detection channel for enhancement of both detection sensitivity and reproducibility. Especially, by using organic solvent as BGE medium, the developed system avoided the interface problem between aqueous running buffer and low‐water‐content chemiluminescence solvent in previous reports. The influencing factors on chemiluminescence signal were optimized using rhodamine 6G as model molecule. The system performance was further investigated in the experiment of separation of hydrophilic rhodamine dyes and analysis of hydrophobic polycyclic aromatic hydrocarbon, providing the detection limit (S/N = 3) of 3.5 nmol/L for rhodamine 123, 6.8 nmol/L for rhodamine 6G, and 60 nmol/L for 1‐aminopyrene, respectively. The experimental results showed the system offered a number of benefits, including compact structure, high sensitivity, good reproducibility, and a wide range of application prospect.     

Performance evaluation of a capillary electrophoresis electrochemical chip integrated with gold nanoelectrode ensemble working and decoupler electrodes

This paper presents a capillary electrophoresis poly(methyl methacrylate) (PMMA) based microchip for electrochemical detection applications featuring embedded gold nanoelectrode ensemble (GNEE) working and decoupler electrodes. In fabricating the microchip, the GNEE films are pressed directly onto the metallic electrode structures using a hot embossing technique, and the microfluidic channels are then sealed using a low-temperature azeotropic solvent bonding method. The detection performance of the microchip is evaluated using dopamine and catechol analytes for illustration purposes. The experimental results show that the GNEE working electrode provides a significantly higher signal response than that obtained from a bulk gold electrode when applied to the detection of dopamine analyte. Compared to a conventional bulk palladium decoupler electrode, the GNEE decoupler electrode reduces both the amplitude of the charge current (3.5 nA vs. 18.7 nA) and the baseline drift at higher separation voltages. The measured baseline current drift for the microchip equipped the proposed GNEE decoupler electrode is around three times smaller than the microchip with the palladium decoupler electrode under the applied separation electric field from 40 V/cm to 240 V/cm. Finally, when detecting a mixture of 1mM dopamine and 1mM catechol, the calculated signal response of the microchip with a GNEE decoupler electrode is approximately five times higher than that obtained from a microchip with a bulk Pd decoupler electrode, resulting in the detection limit of 1 microM for the proposed GNEE-based microchip device. Overall, the results indicate that the proposed capillary electrophoresis-electrochemical detection (CE-ED) microchip with embedded GNEE working and decoupler electrodes provides an ideal solution for sample detection in lab-on-a-chip and micro total analysis applications.     

Simultaneous detection of 19 K‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips

We demonstrate here the power and flexibility of free‐solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild‐type DNA. Here, four large drag‐tags are used to achieve free‐solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K‐ras oncogene. LDR‐FSCE enabled electrophoretic resolution of these 19 LDR‐FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free‐solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR‐FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K‐ras mutations on integrated “sample‐in/answer‐out” devices with amplification, LDR, and detection all on one platform.     

A highly efficient and versatile microchip capillary electrophoresis method for DNA separation using gold nanoparticle as a tag

A highly efficient and versatile method for DNA separation using Au nanoparticles (Au NPs) as a tag based on microchip capillary electrophoresis (MCE) was developed. The thiol-modified DNA-binding Au NPs were utilized as a tag. Target DNA was sandwiched between Au NPs and probe DNA labeled with horseradish peroxidase (HRP). In electrophoresis separation, the difference in electrophoretic mobility between free probe and probe-target complex was magnified by Au NPs, which enabled the resulting mixture to be separated with high efficiency by microchip capillary electrophoresis. Horseradish peroxidase was used as a catalytic label to achieve sensitive electrochemical DNA detection via fast catalytic reactions. With this protocol, 27-mer DNA fragments with different sequences were separated with high speed and high resolution. The proposed method was critical to achieve improved DNA separations in hybridization analyses.     

Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis

An integrated system of a silicon-based microfabricated polymerase chain reaction (microPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was +/-0.3 degrees C using an improved novel "joint-heating" scheme. Thermal cycling was digitally controlled with a temperature accuracy of +/- 0.2 degrees C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by microPCR was achieved in microchip, and application of the integrated microPCR-microchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.